Osteoporosis is characterized by loss of bone mass and strength, leading to fractures that cause pain, loss of independence, and increased mortality. Most FDA-approved drugs for osteoporosis have drawbacks, and only teriparatide (PTH) improves bone formation. We have shown that the nitric oxide (NO)/cGMP/protein kinase G (PKG) signaling pathway mediates anabolic effects of estrogens and mechanical stimulation in bone cells, through increased proliferation and survival of osteoblasts/osteocytes. Nitrates, which generate NO, prevent bone loss in estrogen-deficient rats and increase bone mineral density, cortical thickness, and bone formation markers in post-menopausal women-with changes superior to those reported for PTH. However, nitrates are limited by development of tolerance, induction of oxidative stress, and increased cardiovascular mortality after long-term use. We propose pre-clinical studies testing the effects of cGMP-elevating agents with different mechanisms of action in mouse models of osteoporosis: nitrosyl-cobinamide (NO-Cbi), a novel direct NO- releasing agent that, in contrast to nitrates, does not generate superoxide and may even lower oxidative stress, and two prototype guanylate cyclase (sGC) stimulators, BAY58-2667 and BAY41-8543, which target oxidized or reduced sGC, respectively, to generate cGMP NO-independently. In Specific Aim I, we will test the hypothesis that NO-Cbi and BAY58/41 restore and/or prevent estrogen-deficiency-induced bone loss in mice. We will subject mice to ovariectomy or sham operation, and treat them for 8 weeks with NO-Cbi, BAY58, BAY41, 17-estradiol, or vehicle, starting week 1 or 8 post surgery. We will measure bone architecture and mineral content by micro-CT, bone strength by three-point bending, bone turnover by histomorphometry and serum markers, and osteoblast- and osteoclast-specific gene expression by quantitative RT-PCR. We will examine effects of all three drugs on osteoclast differentiation and function ex vivo. In Specific Aim II, we will test the hypothesis that NO-Cbi and BAY58/41 improve bone quality and bone formation in senescence- accelerated SAMP6 mice, who develop osteoporosis at an early age. We will randomize SAMP6 mice and syngeneic SAMR1 control mice to receive NO-Cbi, BAY58, BAY41, vehicle, or PTH for 4 or 12 weeks, and measure skeletal parameters as in Aim I. Since SAMP6 mice have mesenchymal stem cell deficiencies similar to humans with senile osteoporosis, we will determine effects of NO-Cbi and BAY58/41 on osteogenic differentiation of mesenchymal stem cells ex vivo. By comparing effects of NO-Cbi, BAY58, and BAY41 at doses that increase serum cGMP concentrations to a similar degree, we will determine if NO exerts cGMP- independent effects in bone and assess whether estrogen deficiency and aging produce clinically-significant levels of oxidized sGC. While many pathways are implicated in skeletal maintenance and health, few are as suitable as the NO/cGMP pathway to prevent and treat osteoporosis, because cGMP-elevating agents have recently shown favorable toxicity profiles and beneficial effects in cardiovascular diseases and other disorders.